1. Field of the Invention
The present invention relates to a mutated pyruvate carboxylase gene from Corynebacterium. The mutant pyruvate carboxylase gene encodes a pyruvate carboxylase enzyme which is resistant to feedback inhibition from aspartic acid. The present invention also relates to a method of replacing the wild-type pyruvate carboxylase gene in Corynebacterium with this feedback-resistant pyruvate carboxylase gene. The present invention further relates to methods of the production of amino acids, preferably lysine, comprising the use of this mutant pyruvate carboxylase enzyme in microorganisms.
2. Background Art
Pyruvate carboxylase is an important biotin-containing enzyme found in a variety of plants and animals, as well as some groups of bacteria (Modak, H. V. and Kelly, D. J., Microbiology 141:2619-2628 (1995)). In the presence of adenosine triphosphate (ATP) and magnesium ions, pyruvate carboxylase catalyzes the two-step carboxylation of pyruvate to form oxaloacetate, as shown in the equations below:  ENZ−biotin−CŌ2+Pyruvate→ENZ−biotin+oxaloacetate  (2)
In reaction (1) the ATP-dependent biotin carboxylase domain carboxylates a biotin prosthetic group linked to a specific lysine residue in the biotin-carboxyl-carrier protein (BCCP) domain. Acetyl-coenzyme A activates reaction (1) by increasing the rate of bicarbonate-dependent ATP cleavage. In reaction (2), the BCCP domain donates the CO2 to pyruvate in a reaction catalyzed by the transcarboxylase domain (Attwood, P. V., Int. J. Biochem. Cell. Biol. 27:231-249 (1995)).
In bacteria such as Corynebacterium glutamicum, pyruvate carboxylase is utilized during carbohydrate metabolism to form oxaloacetate, which is in turn used in the biosynthesis of amino acids, particularly L-lysine and L-glutamate. Furthermore, in response to a cell's metabolic needs and internal environment, the activity of pyruvate carboxylase is subject to both positive and negative feedback mechanisms, where the enzyme is activated by acetyl-CoA, and inhibited by aspartic acid. Based on its role in the pathway of amino acid synthesis, and its ability to be regulated, pyruvate carboxylase plays a vital role in the synthesis of amino acids.
Bacteria such as C. glutamicum and E. coli are widely used in industry for the production of amino acids such as L-glutamate and L-lysine. Because of the central importance of pyruvate carboxylase in the production of amino acids, particularly L-glutamate and L-lysine, the exploitation of pyruvate carboxylase to increase amino acid production is of great interest in an industrial setting. Thus, promoting the positive feedback mechanism of pyruvate carboxylase, or inhibiting its negative feedback mechanism, in C. glutamicum or could augment amino acid production on an industrial scale.